The resting muscle, in the absence of any stimulation, is remarkably elastic when stretched and released. When stimulated e.g. by nerve impulses, muscle is activated from the resting state. It develops contractile force, shortens and then relengthens to its original dimension when stimulation ceases. It is well known that muscle develops active force by the cycling of a molecular motor, myosin, to actin filaments in the contractile machinery (sarcomere). Muscle shortens when actin filaments are pulled to slide pass myosin thick filaments, without changing the length of either filament. Very little is known of how contracted muscle restores its length and how resting muscle responds to stretch and compression. It is also unclear how muscle cells manage to control the uniform and precise length of thick and thin filaments when sarcomeres are assembled in developing muscle tissues. Recent studies of muscle cytoskeletal lattices begin to shed lights on both questions. The cytoplasm of striated muscle cells contains, besides actin and myosin filaments, contains at least two interconnected lattices. An intermediate filament lattice envelops and links all sarcomeres to the membrane skeleton (costamere), mitochondria, nuclei, and sarcoplasmic reticulum. Inside the sarcomere, a cytoskeletal matrix consisted of a set of elastic titin filaments and a set of inextensible nebulin filaments provides structural continuity. Both lattices generate restoring force. Active force and elastic force are transmitted through specialized anchor structures of the sarcomere. One important stress-bearing structure is the Z line, a dense and narrow structure that anchors and organizes four major filaments: actin, titin,nebulin and desmin filaments. The Z lines therefore plays a fundamental role in both the structural organization of sarcomere and the transmission of mechanical forces of the sarcomere as well the intermediate filament lattice. Its dense structure however poses technical challenges and the variability of protein composition made it difficult to generalize findings from one muscle to the next. Our projects address the Z line structure and function from several prospective.a. What are the roles of titin, nebulin (skeletal muscles), nebulette (a nebulin-ike protein in the heart) in the assembly and integrity of the Z line in vertebrate muscle? b. What are the composition and structure of the unusually broad Z line of sonic muscle of Midshipman fish? What is its relationship to the anomalous nemaline rod Z bodies found in aging heart muscle, in diseased skeletal muscle known as nemaline myopathy ? The assembly of the titin, nebulin and nebulette into the myofibrils and the Z lines are being studied with fluorescence techniques with either monoclonal antibodies to these proteins, or by the use of fluorescent fusion proteins synthesized within the muscle cells. To identify protein composition, especially the proteins that interact with titin, nebulin and nebulette in the Z line, we are applying both molecular biological methods (yeast two hybrid screening), as well as biochemical techniques techniques to search for interacting proteins. The high-resolution structure of the unusually broad Z band (1 mm, roughly 100 times the wide of vertebrate Z lines) in the sonic muscle of Midshipman fish is being studied by electron microscopy, X-ray diffraction and biochemical methods. The broad Z band is consisted of parallel filaments that are linked by criss crossing linking struts and has a nearly crystalline regularity without the dense amorphous material. X-ray diffraction of live sonic muscle indicated that the linking struts are arranged in a helical fashion around the parallel filaments. Biochemical and immunological staining revealed that actin and alpha-actitnin are major components of the Z band. Interestingly, the Z band are also attachment sites of a very elaborate intermediate filaments lattice that provides the necessary radial force to assemble and maintain the tubular shape of the sonic muscle fiber.These studies are important in the understanding of how contractile machinery assemble during development, how it dissemble during remodeling of muscle tissues, how tension are transmitted during muscle activities and how muscles malfunction in diseases. - Muscle, broad Z-line, nemaline myopathy, sonic muscle, X ray diffraction, electron microscopy, Midshipman fish, crosslinking